INTRODUCTION

Between 1990 and 1999, the average sales of farmed mink and
fox pelts to Western markets were 25.6 and 3.6 million pelts, respectively,
of which 1.8 million mink and 1.9 million fox pelts were produced in Finland
(Anon., 1999a). Compared with the national meat industry, fur farming is a
sufficiently
large business in Finland to be able to utilize all suitable by-products that
become available and therefore, plays an important role in the utilization of
by-products and recycling of nutrients.

Because biologically and hygienically edible parts of cattle
and pigs form only 55 and 63% of the live weight, respectively, great quantities
of different by-products not qualified for human consumption are produced by
meat industry in every country (Bengtsson & Holmqvist 1984). The percentage
of by-products that can be reused as an animal feed varies from one country
and culture to another, but on average it is 40% of carcass weight in cattle,
over 10% in pigs and over 30% in poultry (Miller & De Boer, 1988). Based
on a national meat production of 340 million kg in 1999 and information provided
by a rendering plant (Lahtinen, personal communication), production of
slaughterhouse
by-products (including bones) in Finland exceeded 150 million kg. Fur animals
consumed approximately 95 million kg of those by-products, representing 25%
of total feed for fur animals. In addition, suitable low bone by-products were
also imported, which increased total percentage of by-products in total fur
animal feed to over 30% (Anon., 1999a; Valkosalo, personal communication).

High moisture and nutrient content combined with enzymes and
temperature favoring growth of microbes make slaughter by-products perishable
and if not properly treated, can be even dangerous to the environment and human
health. Due to a continuous offal output from slaughterhouses, but irregular
consumption by fur animals leads to fur farmers having to preserve and store
these materials for several months. Even though ensiling of by-products with
formic acid is well documented, primarily in fox farming (Backhoff, 1943), a
break-through for conservation of offal has not been achieved and great
quantities
of feedstuffs are still stored frozen. Use of formic acid among mink farmers
was considered to be associated with a lowering fur quality and decreased male
reproductive performance. Its use for ensiling was also decreased due to
unreliable
information concerning the toxicity of formic acid, an effective organic acid
preservative (Näveri, 1983). Furthermore, due to a one-feed system and
due to the use of mink as an animal model also for both the blue (
Alopex
lagopus
) and silver (
Vulpes vulpes
) fox, inter-species differences
and performance of foxes with different feeds were not emphasized. Furthermore,
since homogenous fish products better suited large scale ensiling applications,
research was focused on the preservation of these materials (Møller
Jensen
& Jørgensen, 1975).

The possibility to use nonheated low risk slaughterhouse by-products
in fur animal feeds is based on the Council Directive 90/667/EEC (Anon., 1990)
which is implemented in Finland by a ministerial decision on the 7 July 1994.
The decision defines conditions for the use of by-products in fur animal feed.
Provisions of the decision cover transportation and storage including an
official
registration of fur animal feedingstuff processing plants that manufacture fur
animal feed for sale. The basic principle is that the use of by-products must
not threaten the environment, human or animal health. As far as BSE is
concerned,
by Commission decision (2000/418/EC) specified risk material of bovine animals
has to be removed and destroyed after 1 October 2000 in all Member States.
Optionally
to immediate incineration the risk material can be pre-processed in specified
process lines of high risk plants approved for this purpose. It is yet unclear
if this dehydrated product will be allowed in fur animal feed in a Member State.
In Finland BSE specified risk material indicates a removal of approximately
30% of bovine by-products conventionally used as fur animal feed (Lahtinen,
personal communication), which is less than 10% of all meat industry by-products
utilized by fur animals.

In Finland the proper treatment of slaughterhouse by-products
is nationally important because of the very low level of zoonooses in the
country
(Anon., 1999b; Anon., 1999c). In order to maintain this level low future (below
1%), the Finnish Ministry of Agriculture and Forestry created a national
salmonella
control program (Anon., 1994b) which the Commission approved during EU
membership
negotiations in 1994 (94/968/EC). The Finnish Ministry of Agriculture and
Forestry
in 1998 also banned the use of antibiotics in fur animal feed as a preventive
medication (Anon., 1998), highlighting the importance of hygienic quality of
slaughterhouse by-products. There is also special legislation under preparation
in order to prevent the spreading of zoonooses via slaughterhouse by-products
that are fed to fur animals, which will require immediate treatment of
by-products,
in a special section within the slaughterhouse. Heating or ensiling will be
the two alternatives that all by-products designated for fur animal feed have
to go through before leaving the plant.

Salmonella is not a large problem with fur animals, although
pregnant mink and foxes and young fox cubs are vulnerable (Jørgensen,
1985; Kangas, 1982). Instead, the main concern deals with the fact that
contaminated
fur farms are in contact with food producing area. Hence, without being the
initial source of the disease, fur farms may maintain and spread the disease
in a particular area. Furthermore, some farms also produce meat or milk in
addition
to furs. Therefore, it is important that feed manufacturing practices of fur
farming do not contradict, but are in accordance with other fields of animal
production and contribute to the common national goal.

Central substances
of the study

Formic acid

Formic acid (HCOOH) is an organic acid with a molecular weight
of 46.03 and pK
a
of 3.75. It is colourless, transparent liquid with
a pungent odour, an irritant to eyes, skin, and mucous membranes and is miscible
with water. Formic acid occurs naturally in a variety of plants and fruits,
mammalian tissues and insect venoms. Formic acid is a normal constituent of
the body and is metabolically important in the transfer of one-carbon substances
which primarily come from amino acid metabolism, and act as a substrate for
nucleic acids (Mathews & van Holde, 1990; Stryer, 1988). Formic acid is
used in industry during the preparation of a variety of drugs, dyes, and
chemicals.
Formic acid is produced by heating carbon monoxide and sodium hydroxide under
pressure and then treating the resulting sodium formate with sulphuric acid.

Even though some of the antimicrobial action of formic acid
is based on its pH decreasing effect, inhibition of microbes is mainly
associated
with the undissociated molecule under acidic conditions (
Figure 1)
.
Being the strongest organic acid and having a relatively low pK
a
,
formic acid acts both as acidulant and preservative (
Figure 2
). Formic
acid inhibits the growth of yeasts and bacteria and can also eliminate
salmonella
from feeds (Lueck, 1980; Frank, 1994). In animal feeds, formic acid has been
used for a long time to ensile grass for ruminants, but it has now become an
established feed additive for monogastric animals. Formic acid improves the
digestibility of dietary protein and the growth of young piglets, and is
beginning
to replace antibiotic growth promoters in Europe (Partanen et al., 1998;
Partanen
& Mroz, 1999).

Figure 1.
Schematic
representation of the mode of action of preservative acids and their salts.
Undissociated molecules permeate cell membrane, anions accumulate in the
cell and disrupt cell functions. Cell energy sources are depleted in
transporting
protons from cytoplasm in order to re-establish pH-balance.

Formic acid is readily absorbed from
the gut or through skin and mucous membranes. It is also a breakdown product
of methanol and formaldehyde. Regardless of being a natural metabolite, formic
acid is toxic if accumulated in free form in the body. Primates, particularly
humans, are more sensitive to accumulation than nonprimates (Clay et al., 1975).
Symptoms include metabolic acidosis, ocular pathological changes and death
(Frenia
& Schauben, 1992; Clay et al., 1975; McMartin et al., 1977). The rate at
which formic acid is oxidized into CO
2
depends on hepatic levels
of folic acid vitamer THF and on species-specific activities of involving
enzymes
(Johlin et al., 1987) (
Figure 3
). The toxicity of formic acid is based
on its biological activity. It is an inhibitor of cytochrome-oxidase complex
at the terminus of the respiratory chain in mitochondria (Moody, 1991).

Figure 2.
Percentage of formic and benzoic acid molecules that
exist undissociated in media of various pH. The pH where half of the
preservative
is dissociated and half undissociated is indicated by the pKa value, 4.2
and 3.75 for benzoic and formic acids (and their salts), respectively.

Even though formic acid has been known for decades within fur
animal feeding (Backhoff, 1943), its use has also been the subject of some
dispute,
partly due to a lack of knowledge concerning formic acid metabolism
(Näveri,
1983), and partly due to confusion caused by several other concomitant dietary
factors, such as anemiogenic fish and sulphuric acid present in fish silage
(Kangas, 1977; Näveri, 1983) and peroxidized feed fat (Havre et al., 1973).
Furthermore, formic acid was suspected to deliteriously effect reproductive
performance in mink (Näveri, 1983; Näveri, 1984).

Because trimethylamine oxide (TMAO) and its breakdown product,
formaldehyde that exists naturally in anemiogenic fish were identified to render
dietary iron unabsorbable (Ender & Helgebostad, 1968; Costley, 1970), doubts
concerning potential anemiogenic agent were raised (Näveri, 1983). Even
though detrimental effects of formic acid on foxes were not experienced or
verified
with experiments, due to a one-feed system for all fur animal species, use of
formic acid as feed preservative declined. In Denmark, acetic acid was
substituted
for formic acid in sulphur acid-formic acid ensiled fish because of its limited
palatability in mink kits of 4-6 wk of age (Møller Jensen &
Jørgensen,
1975; Jørgensen, 1981). Therefore, one of the objectives of this study
was to investigate and assess fur animal species-specific uses of formic acid.

Figure 3.
Structure of folic acid. A biologically active form
of folic acid is 5,6,7,8-tetrahydrofolate (THF), a highly versatile carrier
of one-carbon units (methyl, methylene, formyl, formimino, methenyl) that
are bonded to the N
5
and N
10
nitrogen atoms. In
the oxidation of formate, THF receives formyl group (catalyzed by N
10
-formylTHF
synthetase, EC 6.3.4.3.) and donates CO
2
(catalyzed by N
10
-formylTHF
dehydrogenase, EC 1.5.1.6.). Because vitamin B
12
is required
for generation of THF from N
5
-methylTHF, a B
12
deficiency
leads to the accumulation of N
5
-methylTHF and hence, to a deficiency
of the functional form of folic acid, THF.

Folic acid

Folic acid, or folates as a generic name, are available from
a large variety of sources such as green leafy vegetables, liver, beans and
fermented dairy products (Vahteristo, 1998). A disease later discovered to be
folic acid deficiency was first described by Wills in 1931 as a "tropical
macrocytic anemia" observed in India, that was often associated with
pregnancy,
and prevented or relieved by extracts of autolyzed yeast and liver (Scott et
al., 1982). In 1940 this unidentified factor was concentrated from spinach and
named according to requirement by
Streptococcus faecalis
. Folates are
necessary for red blood cell formation, metabolism of fats and amino acids,
cell division, protein synthesis, DNA and RNA synthesis, and thus for the growth
and reproduction of all body cells. The importance of folates is based on their
requirement as coenzymes in the transfer and utilization of one-carbon units
in a variety of biosynthetic reactions. Therefore, during periods of rapid cell
regeneration and growth, such as pregnancy and infancy, increased amounts of
folate are required. In addition, folate has further roles in the prevention
of diseases such as neural tube defects and accumulation of homocysteine, a
risk factor for cardiovascular disease (Scott, 1997).

By nomenclature folates are a group of heterocyclic compounds
based on 4-[(pteridin-6-ylmethyl)amino]benzoic acid skeleton conjugated with
one or more, usually 5 to 8, L-glutamic acid residues. Folic acid (
Figure
3)
(mol. wt. 441.4 g) is not present in biological systems but is the form
generally used in pharmaceutical and fortified food products.

Even though the requirement of folic acid in animal feeds is,
markedly lower than 1 mg kg
-1
DM, and it takes several weeks before
deficiency symptoms appear even in growing animals (Thenen & Rasmussen 1978;
Kim et al., 1994), there is evidence that folate status for optimal performance
and development of production animals is achieved with additional
supplementation
as high as above 10 mg kg
-1
feed DM (Matte et al., 1999).

Benzoic acid

Benzoic acid (E 210), C
6
H
5
COOH (mol.
wt. 122.1) is a granular or crystalline powder with a sweet or astringent taste
and is generally known as a preservative. It is mainly used in the form of
sodium
benzoate (E211), C
6
H
5
COONa (mol. wt. 144.1), because the
sodium salt has a higher solubility in water (500g L
-1
) than the
acid (3.4 g L
-1
). Benzoic acid occurs naturally in many acidic fruits
and berries, such as cranberries, plums, cinnamon, and ripe cloves.

Benzoic acid is one of the oldest chemical preservatives used
in the cosmetic, drug and food industry. Sodium benzoate was the first chemical
preservative permitted in food for human consumption in the U.S. in 1908, and
continues to be used in a large number of foods (Jay, 1992). Benzoate inhibits
yeasts more than it inhibits moulds or bacteria. As with other lipophilic acids,
the undissociated form is essential to its antimicrobial activity (
Figure
1
). In undissociated form, benzoic acid and sodium benzoate are soluble
in cell membranes and facilitate proton leakage into cells, increasing cellular
energy requirements to maintain internal pH. Due to a relatively low pK
a
,
4.2, the antimicrobial activity of benzoate is rather low at higher pHs (
Figure
2)
. Instead, alkyl esters of p-hydroxybenzoic acid, parabens, with a pK
a
8.5, are used to extend the activity spectrum to pH 8 (Jay, 1992; Lueck, 1980).
In animal feeds the use of benzoic acid has increased during recent years.
Benzoate
inhibits fungal growth in formic acid treated grass silages (Aronen et al.,
1987). In liquid pig feeds benzoate has been used to inhibit yeast fermentation
(Rantanen, personal communication) and according to Mroz et al. (1998)
Ca-benzoate
in pig feed increases urine acidity and reduces ammonia emissions.

Like other aromatic carboxylic acids, benzoic acid is xenobiotic
and is eliminated from the body, mainly by conjugation with glycine, and to
a lesser extent with glucuronic acid (Bridges et al., 1970). Conjugation occurs
mainly in the liver and kidneys, and the product benzoylglycine, also called
hippuric acid, is excreted in urine (Hutt & Caldwell, 1990) (
Figure 4
).
As an exception to all other studied species, the cat (family
Felidae
)
is rather sensitive to benzoic acid, due to an inability to produce benzoyl
glucuronide (Bedford & Clarke, 1972).

Figure 4
. Congugation of benzoic acid with glycine. In many animal
species conjugation with amino acids is an important route in the
biotransformation
of xenobiotic carboxylic acids before elimination in urine.